Bioassay of brine shrimp lethality and t

Journal of Pharmacognosy and Phytochemistry 2015; 3(6): 43-46

 

E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2015; 3(6): 43-46
Received: 17-01-2015
Accepted: 30-01-2015
Abul Hasanat
Department of Pharmacy,
International Islamic University
Chittagong (IIUC), Chawkbazar,
Chittagong-4203, Bangladesh
Md. Jakaria
Department of Pharmacy,
International Islamic University
Chittagong (IIUC), Chawkbazar,
Chittagong-4203, Bangladesh
Md Ibrahim Tarek
Department of Pharmacy,

International Islamic University
Chittagong (IIUC), Chawkbazar,
Chittagong-4203, Bangladesh
Tanvir Ahmad Chowdhury
Department of Pharmacy,
International Islamic University
Chittagong (IIUC), Chawkbazar,
Chittagong-4203, Bangladesh
Arifujjaman
Department of Pharmacy,
International Islamic University
Chittagong (IIUC), Chawkbazar,
Chittagong-4203, Bangladesh
Md. Sekendar Ali
Department of Pharmacy,
International Islamic University
Chittagong (IIUC), Chawkbazar,
Chittagong-4203, Bangladesh

Correspondence:

Md. Jakaria
Department of Pharmacy,
International Islamic University
Chittagong (IIUC), Chawkbazar,
Chittagong-4203, Bangladesh

Bioassay of brine shrimp lethality and thrombolytic activity
of methanolic extract of Macaranga denticulata leaves
Abul Hasanat, Md. Jakaria, Md Ibrahim Tarek, Tanvir Ahmad Chowdhury,
Arifujjaman, Md. Sekendar Ali
Abstract
The study was aimed to investigate the brine shrimp lethality bioassay and thrombolytic activity of the
methanolic extract of Macaranga denticulata leaves. An in vitro thrombolytic model was used to
evaluate the clot lysis effect of different extracts of M. denticulata along with Streptokinase as a positive
control and distilled water as a negative control. The cytotoxic activity of methanolic extracts of M.
denticulata leaves was evaluated by Brine shrimp lethality bioassay. The brine shrimp lethality bioassay
result was (LC50=82.74 µg/ml) compared with standard vincristine sulphate (LC50=0.839 µg/ml). It has
significant thrombolytic activity (34.77%) compared to standard streptokinase (70%). The results of this
study confirmed that this plant candidate for future research of anticancer and thrombolytic drugs.
Keywords: Brine shrimp, Thrombolytic, Cytotoxic, Clot lysis, Macaranga denticulata.

1. Introduction
Thrombolysis is the breakdown (lysis) of blood clots by pharmacological means. It is
colloquially referred to as ‘clot busting’ for this reason. It works by stimulating fibrinolysis by
plasmin through infusion of analogs of tissue plasminogen activator (tPA), the protein that
normally activates plasmin. In vitro thrombolytic activity of crude extract was enumerated and
was compared with streptokinase, which is a well known anticoagulant used in myocardial
infarction [1]. Brine shrimp lethality bioassay is a bench top bioassay method for evaluating
anticancer, antimicrobial and other pharmacological activity of natural products. Natural
products extracts, fractions or pure compounds can be tested for their bioactivity by this
method [2]. Macaranga denticulata Muell. Arg. (Euphorbiaceae) is a small to medium-sized,
evergreen tree and is a common pioneer species in moist open areas and secondary forests [3].
In the mountains of Northern Thailand, M. denticulata is used as a fallow enriching species by
Karen hill tribe farmers [4]. In folk medicine, traditional healers use fresh or dried leaves of
some Macaranga species to treat swellings, cuts, sores, boils and bruises [5]. A phytochemical
review of literatures indicates the genus Macaranga to be a rich source of the isoprenylated,
geranylated and farnesylated flavonoids and stilbenes. Furthermore, more classes of secondary
metabolites like terpenes, tannins, coumarins and other types of compounds are known to be
isolated from different species of the genus Macaranga. Flavonoids and stilbenes are regarded
as the major constituents and are most likely responsible for most of the activities found in the

plants of this genus. An increasing number of phytochemical studies are being carried out on
plants belonging to the genus Macaranga due to their various traditional uses. Thus, the
isolated natural products from this genus have been reported to display interesting biological
activities including antitumor, antioxidant, antimicrobial and anti-inflammatory [6].
This is a resourceful area of research as many species of Macaranga are used in traditional
medicine as well as exhibits various pharmacological properties while their chemistry
indicates varied chemical structures. The aim of our present work was to investigate the
cytotoxic and thrombolytic activity of methanolic extracts of M. denticulata by using an in
vitro procedure.
2. Materials and Method
2.1 Plant collection: The leaves of M. denticulata were collected from the Chittagong city
area in front of Chittagong Medical college hostel gate of Bangladesh in October, 2014 then
identified by Dr. Sheikh Bokhtear Uddin, Associate Professor, Department of Botany,
University of Chittagong, Chittagong-4331, and Bangladesh.
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Journal of Pharmacognosy and Phytochemistry

 
2.2 Extracts preparation: The collected plant was washed

thoroughly with water and air dried for a week at 35 to 40 °C
and pulverized in electric grinder. The obtained powder was
successively added to methanol with vigorous shaking at 55 to
60 °C temperature. The extracts were made to dry by using
rotary evaporator under reduced pressure. The extract was
preserved at 4 oC for further use.
2.3 Brine shrimp lethality bioassay: For the preparation of
sea water 38 g of sodium chloride was weighed, dissolved in
distilled water to make 1 liter solution and then filtered off to
get clear solution. This simulated sea water was used for
hatching of brine shrimp. The shrimp were allowed for two
days to hatch and mature as nauplii (larvae). In a small beaker,
measured amount of the sample was accurately weighed and
dissolved in DMSO (Dimethylsulfoxide) to give a final
concentration of 5 mg/ml (5 μg/μl). From the test tube
containing brine shrimp nauplii, 6 test tubes were taken for the
sample where each contained 5ml of seawater and 10 nauplii.
These test tubes were marked from 1 to 6 for the sample. To
these test tubes different concentrations (250 μg/ml, 200
μg/ml, 150 μg/ml, 100 μg/ml, 50 μg/ml and 25 μg/ml) of the

sample were added. Then the samples were subjected to brine
shrimp lethality evaluation [7]. In this case, only 50 μl DMSO
was added in 5 ml sea water containing 10 nauplii. No extract
was added to prepare control solution, vincristine sulphate was
used as a standard and LC50 values were calculated.
2.4 Sample preparation: The crude extract was suspended in
10 ml distilled water and shaken vigorously on a vortex mixer.
Then the suspension was kept overnight and decanted to
remove the soluble supernatant, which was filtered through a
filter paper. The solution was then ready for in vitro evaluation
of clot lysis activity.
2.5 Streptokinase (SK) solution preparation: To the
commercially available lyophilized SK vial (PolaminWerk
GmbH, Herdecke, Germany) of 15,00,000 I.U., 5 ml sterile
distilled water was added and mixed properly. This suspension
was used as a stock from which 100 μl (30,000I.U) was used
for in vitro thrombolysis.

2.6 Specimen: Whole blood (5 ml) was drawn from healthy
human volunteers (n=10) without a history of oral

contraceptive or anticoagulant therapy. 500 μl of blood was
transferred to each of the ten previously weighed alpine tubes
to form clots.
2.7 Thrombolytic assay: Experiments for clot lysis were
carried as reported earlier [8]. Venous blood drawn from
healthy volunteers was transferred in different pre-weighed
sterile eppendorf tube (500 μl/tube) and incubated at 37 °C for
45 minutes. After clot formation, serum was completely
removed (aspirated out without disturbing the clot formed).
Each tube having clot was again weighed to determine the clot
weight (clot weight = weight of clot containing tube-weight of
tube alone). Each eppendorf tube containing clot was properly
labeled and 100 μl of plant extract was added to the tubes. All
the tubes were then incubated at 37 °C for 90 minutes and
observed for clot lysis. After incubation, fluid obtained was
removed and tubes were again weighed to observe the
difference in weight after clot disruption. Difference in weight
taken before and after clot lysis was expressed as percentage
of clot lysis. Streptokinase and water were used as positive and
negative control, respectively. The experiment was repeated

several times with the blood samples of different volunteers. %
clot lysis = (Weight of the lysis clot /Weight of clot before
lysis) × 100.
3. Results
3.1 Brine shrimp lethality bioassay: In brine shrimp lethality
bioassay, the methanolic extract of M. denticulata leaves
showed positive result in comparison with the positive control
vincristine sulphate. By plotting the concentration versus
percent (%) of mortality for all test samples showed an
approximate linear correlation. From the graph, the median
lethal concentration (LC50) was determined to check the toxic
level of the extract. The crude extract of M. denticulata leave
showed significant cytotoxic activity against brine shrimp
nauplii and LC50 value was 82.74 µg/ml compared with
standard vincristine sulphate (LC50=0.839 µg/ml). (Table 1 &
Figure 1). DMSO was used as a negative control to validate
the test method.

Table 1: Brine shrimp lethality of Macaranga denticulata
Log

C
1.39
1.69
2
2.17
2.30
2.39

Total
nauplii
10
10
10
10
10
10

No. of nauplii
Dead
02

02
05
06
08
09

No. of nauplii
live
08
07
05
04
02
01

100

% of
mortality
20%

30%
50%
60%
80%
90%

y = 69.185x - 82.678
R2 = 0.9428

90
80
70
%of Mortalty

Concentration
(μg/ml)
25
50
100
150
200
250

60
50
40
30
20
10
0
0.00

0.50

1.00

1.50

2.00

2.50

3.00

Log conce ntraton

Fig 1: Determination of LC50 of methanol extract of Macaranga denticulata
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LC50

82.74
µg/ml

Journal of Pharmacognosy and Phytochemistry

 

3.2 Thrombolytic activity assay: 100 μl Streptokinase as a
positive control (30,000 I.U.) was added to the clots along
with 90 minutes of incubation at 37 °C, showed 70% clot lysis.
Clots when treated with 100 μl sterile distilled water (negative
control) showed only negligible clot lysis (2.8%). The in vitro
thrombolytic activity study revealed that M. denticulata

showed 34.77% clot lysis. The percentage of weight, loss of
clot after application of extract solution was taken as the
functional indication of thrombolytic activity. % Clot lysis
obtained after treating clots with different concentration of the
sample was shown in (Table 2 & Figure 2).

Table 2: Thrombolytic activity of Macaranga denticulata
No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Weight of
empty tube
(A) gm
0.85605
0.81830
0.82150
0.77820
0.80110
0.83070
0.80330
0.80475
0.80875
0.80925

Weight of
clot tube with
(B) gm
1.2178
1.1299
1.1881
1.1531
1.08195
1.08135
1.0316
1.0233
1.0399
1.0646

Weight of
clot (C)
(B-A) gm
0.36175
0.3116
0.3666
0.3749
0.28085
0.25065
0.2283
0.21855
0.23115
0.25535

Weight of tube
with clot after
lysis (D) gm
1.00115
1.09205
1.05705
0.97515
1.005
1.00075
0.9693
0.92755
0.9539
1.0019

Weight of
lysis (E)
(B-D)
0.21665
0.03785
0.13105
0.17795
0.07695
0.08085
0.0623
0.09575
0.086
0.0627

% of clot
lysis

Average % of clot lysis

59.89
12.15
35.75
47.47
27.40
32.26
27.29
43.82
37.15
24.56

34.77%

80%
70%
60%

% of activity

50%
40%
..
30%
20%
10%
0%
Streptokinase

Macaranga deniculata

Water

Fig 2: Clot lysis by streptokinase, Macaranga denticulata and water

4. Discussion
This study evaluated the brine Shrimp lethality and
thrombolytic activity bioassay of methanolic extract of M.
denticulata. the herbal preparations are used since ancient
times for the treatment of diseases. Phytopharmacological and
phytochemical evaluation lead to drug discovery. About 30%
of the pharmaceuticals are prepared from plants worldwide [910]
. A number of studies have been conducted by various
researchers to find out the herbs and natural food sources and
their supplements having thrombolytic (anticoagulant and
antiplatelet) effect and there is evidence that consuming such
food leads to prevention of coronary events and stroke[11-14].
Although there are several thrombolytic drugs, including those
obtained by recombinant DNA technology, but side effects
related to some of these drugs that lead to further
complications have been reported [15-18]. Brine shrimp lethality
bioassay is an easy and straight forward bench top screening
method for predicting important pharmacological activities
like enzyme inhibition, ion channel interference, antimicrobial
and cytotoxic activity [19-21]. The extract showed LC50 at a very
low concentration with very quick response indicating that the
extract is significantly potent. Ideally, any agent useful in the
treat men to of cancer should not be toxic to normal cell.
However, in reality, anticancer agents are often toxic to normal
cells, particularly towards rapidly growing cells [22]. It is

necessary to test this extract in low concentration to evaluate
its potency and also against various cancer cell lines as normal
cell lines to justify the potential to further investigate this plant
for anticancer activity. Further investigation is required to find
the responsible compound(s) for the cytotoxic activity
observed for M. denticulata. In the thrombolytic bioassay
result suggested that the extract showed very potent activity.
The plant can be evaluated to further research for thrombolytic
activity to a specific disease.
5. Conclusion
From our bioassay, we concluded that M. denticulata has got
the potential as a candidate for future thrombolytic agent. It
can also be investigated as a possible source of antitumour
drugs. This is only a preliminary study and to make final
comment the extract should thoroughly investigated be
phytochemically and pharmacologically to exploit their
medicinal and pharmaceutical potentials. This study may be
helpful for further research works.
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